Project Summary: The Role of the Mutant p53-PARP-MCM Pathway in Triple Negative Breast Cancer Mutant p53 (mtp53) is found in 80% of triple negative breast cancers (TNBCs). There is a need to increase understanding of mtp53 signaling in TNBC in order to determine strategies to detect and treat the disease. TNBCs are more common in African American women than in white women and therefore the proposed work will benefit all women with TNBC including the AA over-represented population. This application proposes to determine the biological role of TNBC-associated gain-of-function oncogenic mtp53 R273H that is associated with PARP1 (herein referred to as PARP) protein, MCM2-7 helicases and replicating DNA. The p53 Arg 273 substituted with His, and other hotspot p53 missense mutant proteins, are well known to function as oncogenic proteins in TNBCs, but their functions outside of acting as aberrant transcription co-factors are not clearly understood. The R273H mtp53 is present in high levels on chromatin in many TNBCs and works as a co-factor to activate the transcription of sterol biosynthesis genes and chromatin remodeling factors, but incredibly R273H also co-associates with PARP and MCMs on replicating DNA. Our studies have uncovered this novel replication- associated pathway and we call it the mtp53-PARP-MCM chromatin axis. Our preliminary and published data reports that knockdown of mtp53 R273H reduces chromatin associated MCM2-7 and PARP. Knockdown of mtp53 in this setting decreases combined PARP inhibitor (PARPi) talazoparib plus DNA damaging agent temozolomide-mediated cell death, demonstrating that mtp53 expression influences the sensitivity of cancer cells to synergistic PARPi plus DNA damage mediated cell killing. Our preliminary and published data showed nuclear mtp53 R273H co-associates with PARP and MCMs and mtp53 R273H directly associates with EdU at nascent replication forks. Furthermore, we detected high mtp53 R273H, high PARP protein, and high PARP enzymatic activity in patient derived xenograft tissue expressing R273H mtp53. We also detected high co-association of mtp53 and PARP in TNBC in a preliminary tumor microarray screen as well as through analysis of The Cancer Genome Atlas reverse phase protein data sets. As a result, we consider understanding the biology of chromatin-associated mtp53-PARP-MCM complexes on replicating DNA to be an area of inquiry that will have a sustained long-term influence on our understanding, detection, and treatment choices for TNBC in diverse populations of women. The mtp53-PARP-MCM axis in TNBC is an innovative pathway and elucidating the mechanistic relationship of the proteins working together in replication-associated activities will help us to better understand the disease. Our hypothesis is that stable mtp53 interacts with replicating DNA during replication stress using amino acids from its carboxy-terminal regions. As a consequence, the mtp53-PARP-MCM axis is engaged allowing increased recruitment of protein complexes and sensitivity to PARP inhibitors combined with DNA damaging agents. We will test this hypothesis by the following three aims. Aim 1: Elucidate the role and mechanism of the TNBC-associated oncogenic mtp53- PARP-MCM axis. Aim 2: Increase our understanding of co-expression of mtp53 and PARP using novel imaging platforms in xenograft mouse models and screening of multiethnic human breast tissue microarrays to benefit all women with TNBC. Aim 3: Validate the driver role of the mtp53-PARP-MCM axis influencing PARPi outcomes in orthotopic mouse models. Completing this work will provide mechanistic insights on how mtp53 cross-talks with PARP and MCM proteins on chromatin in cell culture and xenograft models, and will inform the potential of using novel imaging agents to detect nuclear co-expression of mtp53 and PARP as informative biomarkers for TNBC.